The 2,4-thiazolidinedione (TZD) ring is found in currently marketed and investigational therapeutic agents. This is a potential human health concern since hepatotoxicity was reported in some patients taking TZD ring containing drugs. An example of this is troglitazone, which was formerly used in the treatment of type II diabetes. Animal models did not predict troglitazone-induced liver damage and the mechanism of this toxicity in humans is unknown. Biotransformation by cytochrome P450 (CYP) isozymes is critical for the hepatotoxicity of another TZD derivative, known as 3-(3,5- dichlorophenyl)-2,4-thiazolidinedione (DCPT), in rats. DCPT may be a useful model compound to evaluate TZD ring-induced liver damage. However, the correlation between metabolism of TZD ring containing compounds and hepatotoxicity, such as DCPT, has not been thoroughly studied in human systems. This project is designed to examine the hypothesis that a CYP-derived metabolite of DCPT and other TZD derivatives will exert cytotoxicity in a human cell line. The experimental approach described below was chosen because it is amenable to involvement of students. Specific Aim 1 is designed to evaluate the feasibility of using HepG2 hepatoma cells, transfected with various human CYPs, to further explore the relationship between TZD rings and toxicity. Transfected cells will be fully characterized for the presence of the CYPs, and will then be incubated with DCPT or other TZD derivatives. Cytotoxicity will be assessed by measuring cellular metabolism and DNA content. Once suitable HepG2 transfectant(s) are found, they will be used in Specific Aim 2 to explore the mechanism of cytotoxicity. The following endpoints will be used to in an effort to discriminate between necrotic and apoptotic cell death pathways: (a) cell membrane permeability, (b) cellular ATP content, (c) nuclear morphology and (d) staining with annexin V-FITC/propidium iodide. Finally, the possibility that CYPs may convert DCPT or similar compounds to potentially toxic "reactive intermediates" will be explored in Specific Aim 3. Using the HepG2 transfectants, experiments will be conducted to see if glutathione (GSH) can detoxify any putative reactive metabolites. In addition, liquid chromatography-mass spectrometry will be used to characterize the structure(s) of any GSH conjugates that may be formed in the cells. This could provide information about the structures of any reactive metabolites that are produced from the test compounds. Collectively, the studies described in this proposal should provide useful information about the metabolism and toxicity of TZD derivatives. PUBLIC HEALTH RELEVANCE: The 2,4-thiazolidinedione (TZD) ring, found in current and prototype drugs, may be a factor in hepatotoxicity (liver damage) in some patients who take these therapeutic agents. This project is designed to develop an in vitro, non-animal based system to investigate the relationship between TZD rings and hepatotoxicity. An understanding of this connection may lead to development of better and safer drugs that contain TZD rings.